The present invention relates to an apparatus and method for use in identifying an oligomeric compound (or molecule) that is synthesized into other chemical compounds, particularly those compounds such as pharmaceuticals and biochemicals, by the techniques of combinatorial chemistry. The identification of the oligomeric is performed by identifying the chemical reaction exposure and assay steps that the oligomeric compound has undergone in its synthesis.
The tracking of the progress of chemical reactions through long sequences of synthesis operations is a matter of ever increasing importance in the development and production of new biochemicals and pharmaceuticals.
One particular example of sequences of reactions of the above described type is known as combinatorial synthesis. Combinatorial synthesis may be defined as the generation of a large and diverse series of compounds by the parallel application of different sequences of synthetic reaction steps, or the addition of different functional chemical groups to a population of previously synthesized molecules. These molecules may be peptides, nucleotides, small organic molecules, etc. These large and diverse series of compounds are referred to as combinatorial libraries. Combinatorial libraries are made by forming all possible combinations of a series of sets of precursor molecules, and applying the same sequence of reactions to each combination. Developments in automation have made it possible to synthesize thousands to billions of distinct compounds in parallel. Early work on processes of this type are described in U.S. Pat. No. 4,833,092 (Geysen), which discusses the parallel synthesis of multiple peptides for use in antibody recognition studies.
Research involving pharmaceuticals and biochemicals is particularly well suited for the use of combinatorial synthesis. An objective of combinatorial synthesis is to create libraries of potential pharmaceutically active compounds. Each of these library compounds can then be examined to determine their binding affinity for a target molecule, where in a pharmaceutical application that binding affinity is characterized as bioactivity. After an assay step in which where there is a binding reaction, the identity or reaction history of a library compound must be determined. This is particularly important because the complex synthesis of biomolecules and potential pharmaceuticals involve a large number of reaction steps.
Furthermore, the techniques described herein can be well utilized in creating libraries for many types of complex molecular syntheses, such as lipids used in detergents, heterocyclic compounds used in fuels, and long chain polymers used in materials. One skilled in the art would recognize that the apparatus and method described herein has utility in identifying each of the compounds in combinatorial libraries that are the products of synthesis reactions used in various areas of commerce.
There are at least three approaches to the determination of these novel compounds: direct structural determination; geometric separation of synthetic and assay steps in defined areas; and identification of the initial oligomeric compound and the chemical reaction sequence that the initial oligomeric compound has undergone by attaching these compounds to tagged beads that record this information by chemical, physical, or electronic; or alternatively transmit a distinct identification (ID) code for each bead. Beads of this type are ordinarily used in large numbers that are processed simultaneously in the same reaction vessel. During a series of synthetic steps, these beads are combined, separated and recombined in new combinations as they pass through a series of reaction vessels to produce a number of different end products simultaneously.
Direct structural determination is often not desirable, because each different molecule is often synthesized in quantities too small for structural determination via standard techniques such as nuclear magnetic resonance spectroscopy, mass spectroscopy or chromatographic fragment identification.
Geometric separation of synthetic and assay steps is exemplified by a chip developed by Fodor et al., and described in Science 251, p. 767 (1991). These investigators developed a process to perform a photochemical linkage of peptides and nucleotides on a planar substrate. Different molecules are made in checkerboard pattern on a board. Assay of receptor binding occurs in place on the board. However, this device is not generally applicable to the standard steps used to synthesize organic pharmaceuticals, many of which are not light driven.
The third identification approach in combinatorial synthesis is to tag small beads. Each tagged bead either includes an identification code distinct from each of the other beads, or includes a recording apparatus that records the sequence of reaction it undergoes during the combinatorial process. The tracking process includes a terminal apparatus that examines the beads and determines information about the chemical reactions to which each bead has been exposed. When this information is analyzed, the reaction history of a complex of series of reactions is determinable. Combinatorial bead libraries comprise combinatorial libraries wherein the generated compounds are attached to beads. Combinatorial chemical bead libraries for drug discovery applications comprise 10.sup.2 -10.sup.6 beads. Future libraries may be substantially larger. Thus, a requirement of a bead identification method is high information readout rate.
In most combinatorial synthetic systems that have been implemented to date, information about each synthetic step that the bead has been, or is to be exposed to, is encoded in the bead either prior to placement in a reaction vessel where a particular synthetic step is to be carried out, in the reaction vessel, or after removal from the reaction vessel, and before subsequent placement in a next reaction vessel. At the completion of a given synthetic protocol, chemicals synthesized on the beads are assayed. Information encoded in those beads whose attached synthesized chemicals perform well in the assay is decoded. That information provides the reaction history of each particular bead to enable replication of its attached chemical.
An alternative tagging approach is to tag beads physically with a distinct identification number or code that is predetermined (permanent) during the reaction process. Read out of the bead identification number or code is accomplished by electrical or alternatively electromagnetic radiation transmission. In this approach, each bead is interrogated as to its identification number or code and location prior to, during, and/or after each synthetic reaction, and that information is recorded. That interrogation and recording is accomplished by means of a terminal apparatus computing device because the high readout rate requirement imposed by a relatively large number of beads and a relatively brief available read out time. At the completion of a given synthetic protocol, chemicals synthesized on those beads are assayed. Tag identification of beads on which the chemicals that perform well in the assay are is then determined, and the bead is uniquely identified and correlated with the stored reaction step information to provide the reaction history of each bead, and thus enable replication of the attached oligomeric compound.
There are many possible tagging systems. Beads may be tagged chemically or electronically during the course of a combinatorial synthesis, and alternatively may be tagged with a predetermined (or permanent) electrical, electromagnetic, or physical Identification number or code.
Chemical tags have been implemented by multiple groups and companies. In these protocols, distinct chemical moieties that represent, or code for each step in the synthetic process are added to the bead. After each synthetic and labeling step, the beads are combined, resorted, and a new step in the process is performed. At the end of the process, after the binding assay, beads that have successfully bound the target molecule are separated. The chemical tag is then decoded. This approach is exemplified by the approach of Brenner and Lerner, Proc. Natl. Acad. Sci., U.S.A., 89, pp. 5381-5383 (1992), where the theoretical synthesis of oligonucleotides is used to code for the sequential addition of amino acids to growing peptide chains. This approach is advantageous because the art of oligonucleotide synthesis is well established. This approach suffers, however, from the fact that decoding of the tag requires a slow and complex multi-step procedure and the fact that not all of the original synthetic steps are compatible with the stability of the nucleotide bond.
Another approach to chemical tagging is that described in previously cited U.S. Pat. No. 5,565,324 (Still et al.) used extensively by Pharmacopeia, Inc., in their combinatorial syntheses. Here a variety of different tags are used for various synthetic steps. Release and chemical identification tags are used to fingerprint the specific chemical synthesized. This approach has the advantage that the tags do not have to be bound in an oligomeric or polymeric form. Therefore, the tags do not have to be sequenced. This approach has the disadvantage that the tags must be robust to all process steps and that the tags must be chemically analyzed upon completion of a binding assay.
Prior to the present invention, there has been developed a combinatorial bead which is labeled electronically, via an encapsulated electronic system. This bead, which is now being commercialized by Irori, Inc., was developed by K. C. Nicolaou and Xiao-Yi Xiao and is described in Chem. Int. Ed. Engl., 34, p. 2289 (1995), and also disclosed in International Application Number: PCT/US96/06145. In this application, the chip includes encapsulated memory devices associated or coated directly with derivatized polymer during combinatorial synthesis. The chip encodes information about the synthetic pathway, including the reagents used and the conditions of synthesis. The device can then report this information to a receiver via a radio frequency link. A related approach has been developed by Edmund Moran at Ontogen Corp. and described in J. Am. Chem. Soc., 117, p. 10787 (1995).
The Ontogen and Irori systems make use of RF transponder and readout technology developed by Bio Medic Data Systems and Avid Corporation, among others. These systems provide RF tagging capsules and tracking systems for animal and equipment monitoring. One system of this type is described in U.S. Pat. No. 5,252,962 (Urbas) and includes a passive transponder which has a receive antenna for input signals. A frequency generator that receives the input signal and outputs a data carrier signal having a frequency independent of the input signal frequency. A programmable memory and thermistor are provided to produce user identification (ID) data and temperature data which are combined with an output signal. Other systems of this type are described in U.S. Pat. No. 5,351,052; U.S. Pat. No. 5,214,409; U.S. Pat. No. 5,257,011; and U.S. Pat. No. 5,266,926.
The IRORI Quantum Microchemistry system uses an encapsulated RF EEPROM memory device which has dimensions of 8.times.1.times.1 mm. The encapsulation consists of TentaGel-like polymer beads carrying an acid-clevable linker to nucleate synthesis of a peptide library of compounds, and a chemically inert, surrounding porous support. Internally the bead includes a memory and temperature sensing unit encapsulated in glass. The memory device is completely passive. Power is provided to the RF systems via magnetic inductive coupling to an antenna coil wound around a ferrite core. The antenna is encapsulated with Rectifier/ regulator, frequency generator and data logic, electrically erasable logic and thermal sensing chips.
The primary size limitation in the RF chip approach is related to providing power to the chip, and placing transmission antennas directly and inexpensively on the RF chip. Off chip batteries can be used or magnetically coupled power as in the previously described embodiments are possible. To date, planar antennas have been inefficient and cannot couple enough energy into the chip to power the device. Recent advances in planar antenna fabrication technology hold the promise of the availability of integration of transmission antennas that operate at up to 40 Ghz with CMOS circuitry. Work of this type is described in the Nov. 25, 1996 issue of Electronic Engineering Times. However, even with such antennas, it is impractical for chips to be powered by RF or microwave beams because such chips cannot be made small enough to be useful.
All of the above-described devices have limitations that restrict their usefulness. One major limitation of these devices is a lack of durability associated with their size and weight. If, for example, the beads include an on-bead power source such as a battery, they will be so large and heavy that they are unlikely to long survive the jostling associated with movement through a sequence of reaction vessels. Even if the beads include an off-chip power source, the on-chip energy coupling devices such as the ferrite core and associated pickup coil again make the bead so heavy that it is unlikely to long survive processing through a sequence of reaction vessels. Attempts to replace such cores and coils with microwave power receiving antennas result in inadequate received power levels even when those antennas are made so large as to make the associated chips unwieldy.
Another approach to combinatorial tagging is to physically mark each bead with an encoding mark at each synthetic step in the combinatorial process. This approach is exemplified by the "optical spectral hole burning" system described in IRORI PCT filing number PCT/US96/06145. Optical spectral hole burning uses an intense laser beam to burn a hole in the absorption spectral profile of a suitable material. Spectral hole burning may be present only during the laser pulse, or be a long lasting phenomenon. For use in a bead marking system the absorption hole must be long lasting, and insensitive to all temperature excursions that the bead may undergo during synthesis. A spectral hole burning system for use as an optical memory is described in U.S. Pat. No. 5,136,572. The key to a long lasting realizable optical spectral hole burning system is the availability of a material with an inhomogeneously broadened absorption spectra that supports the availability of many spectral holes at the same spatial position. Materials exist that satisfy these conditions at low temperature, but not at room or elevated temperatures. A few spectral holes may be generated at room temperature. Thus synthetic information must be encoded in a geometrically defined series of independent spectral holes. This is not a robust encoding scheme, and it is not clear whether the spectral holes are stable to all synthetic conditions.
Many combinatorial synthetic protocols use between 10.sup.2 -10.sup.6 beads. It is advantaged to provide each bead with its own predetermined (or permanent) ID tag. The tagging mechanism may be electromagnetic, or physical. Examples of physical tags include bar codes and alphanumeric codes as described in IRORI PCT filing number PCT/US96/06145.
Specifically, the IRORI PCT captures the concept of using alphanumeric marks and bar codes as ID codes for tracking combinatorial beads, claiming an alphanumeric code or a bar code, as well as tagging a "matrix" with an identifying mark. Marking synthetic matrices with bar codes or alphanumeric markings requires an imaging readout of the code before each synthetic step. This is not always practical with small beads that are not completely oriented, and information extraction from an image may take a substantial time period. Other tagging schemes disclosed here are more advantaged.
In view of the foregoing, it is seen that a need exists for a mobile tracking device which is small and inexpensive to produce, which does not require the use of batteries or radio or microwave links, is easy and fast to read, and yet which is both durable and able to perform all of the functions required of it during its use in a combinatorial synthesis apparatus.
The invention of this patent is a bead tagging device that emits an electromagnetic wave whose spectrum is alternatively a distinct combination code, a distinct permutation code, and a distinct permutation code whose separate temporal components may be a combination code; and whose spectrum therefore distinctly identifies each bead. The invention of this patent is furthermore a terminal apparatus that receives that spectrum and distinctly identifies each bead. The invention of this patent is furthermore a method for bead identification that uses the bead tagging device and terminal apparatus of this invention. The precise meaning of a combination code and a permutation code with respect too this invention are described presently.
A combinatorial chemistry bead ID Tag with an electromagnetic combination, permutation, or combined permutation/combination identifying code should satisfy the following conditions: 1) the frequency of each spectral component in the tag must be distinct from all other spectral; 2) the efficiency of emission of each spectral component must be high enough to ensure reliable detection, with a high Signal/Noise ratio; 3) the tagged bead should be small, less than 2 mm in its largest physical dimension; 4) the entire ID code for each bead must be transmitted and verified in less than 50 milliseconds; 5) the tag system should allow spatial localization of ID code readout, consistent with high speed identification and sorting; 6) the stimulus signal should be able to be applied to all orientations of the bead that the bead may acquire during the ID tag reading process; 7) the tag emission spectral signature should be non-directional, or controllable to allow reliable readout or be consistent with a readout system that acquires signals from all relevant directions of emission; 8) the stimulus and ID spectral signatures must not significantly interfere with, or cause damage to, the growth of, or assay of, molecules on the combination synthetic bead; 9) the stimulus and ID spectral signatures must not be significantly absorbed by the fluid, or vessel in which the bead is disposed during readout; and 10) the tag system must be stable under all temperature, pressure and fluidic environments the bead may encounter during synthesis, readout, assay, and storage.